TY - JOUR
A1 - Deutschmann, Claudia
A1 - Roggenbuck, Dirk
A1 - Schierack, Peter
A1 - Rödiger, Stefan
T1 - Autoantibody testing by enzyme-linked immunosorbent assay-a case in which the solid phase decides on success and failure
JF - Heliyon
N2 - Background: The enzyme-linked immunosorbent assay (ELISA) is an indispensable tool for clinical diagnostics to identify or differentiate diseases such as autoimmune illnesses, but also to monitor their progression or control the efficacy of drugs. One use case of ELISA is to differentiate between different states (e.g. healthy vs. diseased). Another goal is to quantitatively assess the biomarker in question, like autoantibodies. Thus, the ELISA technology is used for the discovery and verification of new autoantibodies, too. Of key interest, however, is the development of immunoassays for the sensitive and specific detection of such biomarkers at early disease stages. Therefore, users have to deal with many parameters, such as buffer systems or antigen-autoantibody interactions, to successfully establish an ELISA. Often, fine-tuning like testing of several blocking substances is performed to yield high signal-to-noise ratios.
Methods: We developed an ELISA to detect IgA and IgG autoantibodies against chitinase-3-like protein 1 (CHI3L1), a newly identified autoantigen in inflammatory bowel disease (IBD), in the serum of control and disease groups (n = 23, respectively). Microwell plates with different surface modifications (PolySorp and MaxiSorp coating) were tested to detect reproducibility problems.
Results: We found a significant impact of the surface properties of the microwell plates. IgA antibody reactivity was significantly lower, since it was in the range of background noise, when measured on MaxiSorp coated plates (p < 0.0001). The IgG antibody reactivity did not differ on the diverse plates, but the plate surface had a significant influence on the test result (p = 0.0005).
Conclusion: With this report, we want to draw readers' attention to the properties of solid phases and their effects on the detection of autoantibodies by ELISA. We want to sensitize the reader to the fact that the choice of the wrong plate can lead to a false negative test result, which in turn has serious consequences for the discovery of autoantibodies.
KW - biochemistry
KW - coatings
KW - surface chemistry
KW - immunology
KW - proteins
KW - laboratory medicine
KW - clinical research
KW - enzyme-linked immunosorbent
KW - assay
KW - biomarker discovery
KW - reproducibility
KW - solid-phase
KW - autoantibody
Y1 - 2020
U6 - https://doi.org/10.1016/j.heliyon.2020.e03270
SN - 2405-8440
VL - 6
IS - 1
PB - Elsevier
CY - London [u.a.]
ER -
TY - GEN
A1 - Choi, Youngeun
A1 - Schmidt, Carsten
A1 - Tinnefeld, Philip
A1 - Bald, Ilko
A1 - Rödiger, Stefan
T1 - A new reporter design based on DNA origami nanostructures
for quantification of short oligonucleotides using microbeads
T2 - Postprints der Universität Potsdam : Mathematisch-naturwissenschaftliche Reihe
N2 - The DNA origami technique has great potential for the development of brighter and more sensitive reporters for fluorescence based detection schemes such as a microbead-based assay in diagnostic applications. The nanostructures can be programmed to include multiple dye molecules to enhance the measured signal as well as multiple probe strands to increase the binding strength of the target oligonucleotide to these nanostructures. Here we present a proof-of-concept study to quantify short oligonucleotides by developing a novel DNA origami based reporter system, combined with planar microbead assays. Analysis of the assays using the VideoScan digital imaging platform showed DNA origami to be a more suitable reporter candidate for quantification of the target oligonucleotides at lower concentrations than a conventional reporter that consists of one dye molecule attached to a single stranded DNA. Efforts have been made to conduct multiplexed analysis of different targets as well as to enhance fluorescence signals obtained from the reporters. We therefore believe that the quantification of short oligonucleotides that exist in low copy numbers is achieved in a better way with the DNA origami nanostructures as reporters.
T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 705
KW - nucleic-acids
KW - hybridization
KW - microrna
KW - flourescence
KW - biomarkers
KW - platform
KW - particle
KW - binding
KW - array
KW - gene
Y1 - 2019
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-428271
SN - 1866-8372
IS - 705
ER -
TY - JOUR
A1 - Choi, Youngeun
A1 - Schmidt, Carsten
A1 - Tinnefeld, Philip
A1 - Bald, Ilko
A1 - Rödiger, Stefan
T1 - A new reporter design based on DNA origami nanostructures for quantification of short oligonucleotides using microbeads
JF - Scientific Reports
N2 - The DNA origami technique has great potential for the development of brighter and more sensitive reporters for fluorescence based detection schemes such as a microbead-based assay in diagnostic applications. The nanostructures can be programmed to include multiple dye molecules to enhance the measured signal as well as multiple probe strands to increase the binding strength of the target oligonucleotide to these nanostructures. Here we present a proof-of-concept study to quantify short oligonucleotides by developing a novel DNA origami based reporter system, combined with planar microbead assays. Analysis of the assays using the VideoScan digital imaging platform showed DNA origami to be a more suitable reporter candidate for quantification of the target oligonucleotides at lower concentrations than a conventional reporter that consists of one dye molecule attached to a single stranded DNA. Efforts have been made to conduct multiplexed analysis of different targets as well as to enhance fluorescence signals obtained from the reporters. We therefore believe that the quantification of short oligonucleotides that exist in low copy numbers is achieved in a better way with the DNA origami nanostructures as reporters.
KW - nucleic-acids
KW - hybridization
KW - microrna
KW - flourescence
KW - biomarkers
KW - platform
KW - particle
KW - binding
KW - array
KW - gene
Y1 - 2019
U6 - https://doi.org/10.1038/s41598-019-41136-x
SN - 2045-2322
IS - 9
PB - Macmillan Publishers Limited
CY - London
ER -
TY - JOUR
A1 - Schmidt, Carsten
A1 - Schierack, Peter
A1 - Gerber, Ulrike
A1 - Schroeder, Christian
A1 - Choi, Youngeun
A1 - Bald, Ilko
A1 - Lehmann, Werner
A1 - Rödiger, Stefan
T1 - Streptavidin homologues for applications on solid surfaces at high temperatures
JF - Langmuir
N2 - One of the most commonly used bonds between two biomolecules is the bond between biotin and streptavidin (SA) or streptavidin homologues (SAHs). A high dissociation constant and the consequent high-temperature stability even allows for its use in nucleic acid detection under polymerase chain reaction (PCR) conditions. There are a number of SAHs available, and for assay design, it is of great interest to determine as to which SAH will perform the best under assay conditions. Although there are numerous single studies on the characterization of SAHs in solution or selected solid phases, there is no systematic study comparing different SAHs for biomolecule-binding, hybridization, and PCR assays on solid phases. We compared streptavidin, core streptavidin, traptavidin, core traptavidin, neutravidin, and monomeric streptavidin on the surface of microbeads (10-15 mu m in diameter) and designed multiplex microbead-based experiments and analyzed simultaneously the binding of biotinylated oligonucleotides and the hybridization of oligonucleotides to complementary capture probes. We also bound comparably large DNA origamis to capture probes on the microbead surface. We used a real-time fluorescence microscopy imaging platform, with which it is possible to subject samples to a programmable time and temperature profile and to record binding processes on the microbead surface depending on the time and temperature. With the exception of core traptavidin and monomeric streptavidin, all other SA/SAHs were suitable for our investigations. We found hybridization efficiencies close to 100% for streptavidin, core streptavidin, traptavidin, and neutravidin. These could all be considered equally suitable for hybridization, PCR applications, and melting point analysis. The SA/SAH-biotin bond was temperature sensitive when the oligonucleotide was mono-biotinylated, with traptavidin being the most stable followed by streptavidin and neutravidin. Mono-biotinylated oligonucleotides can be used in experiments with temperatures up to 70 degrees C. When oligonucleotides were bis-biotinylated, all SA/SAH-biotin bonds had similar temperature stability under PCR conditions, even if they comprised a streptavidin variant with slower biotin dissociation and increased mechanostability.
Y1 - 2020
U6 - https://doi.org/10.1021/acs.langmuir.9b02339
SN - 0743-7463
VL - 36
IS - 2
SP - 628
EP - 636
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Czarnecka, Malgorzata
A1 - Weichelt, Ulrike
A1 - Rödiger, Stefan
A1 - Hanack, Katja
T1 - Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA
JF - International Journal of Molecular Sciences
N2 - The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.
KW - antibody
KW - camelid antibody
KW - heavy-chain-only antibody
KW - miRNA
KW - nucleic acids
KW - novel biomarkers
Y1 - 2022
U6 - https://doi.org/10.3390/ijms23116275
SN - 1422-0067
VL - 23
SP - 1
EP - 18
PB - MDPI
CY - Basel, Schweiz
ET - 11
ER -
TY - GEN
A1 - Czarnecka, Malgorzata
A1 - Weichelt, Ulrike
A1 - Rödiger, Stefan
A1 - Hanack, Katja
T1 - Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA
T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
N2 - The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.
T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1274
KW - antibody
KW - camelid antibody
KW - heavy-chain-only antibody
KW - miRNA
KW - nucleic acids
KW - novel biomarkers
Y1 - 2022
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-569142
SN - 1866-8372
SP - 1
EP - 18
ER -